Methanol-assimilating yeasts are those capable of growing on methanol as a sole carbon source. In the first reaction of the methanol metabolism in the methanol-assimilating yeasts, formaldehyde and hydrogen peroxide are produced from methanol and oxygen by alcohol oxidase.
The hydrogen peroxide produced is decomposed by catalase into water and oxygen. The formaldehyde, on the other hand, is oxidized into carbon dioxide by formaldehyde dehydrogenase, S-formylglutathione hydrolase and formate dehydrogenase, and NADH produced in these reactions is utilized as an energy source for cells. Simultaneously, the formaldehyde is condensed with xylulose-5-phosphate by dihydroxyacetone synthase to be converted into glyceraldehyde-3-phosphate and dihydroxyacetone which are then converted to cell constituents via the pentose phosphate pathway.
When the methanol-assimilating yeasts are cultured in the presence of methanol, the above-mentioned alcohol oxidase, dihydroxyacetone synthase and formate dehydrogenase are produced in significant amounts and their contents reach about 40% of the intracellular soluble proteins.
Because a large scale cultivation of the methanol-assimilating yeasts can be done with inexpensive methanol as described above, and because they possess methanol inducible promoters with a strong transcriptional activity not observed in other yeasts, the methanol-assimilating yeasts can be considered to be yeasts suitable for a heterologous gene expression system.
Candida boidinii is one of the methanol-assimilating yeasts, and this yeast is used for studying a method of expressing a heterologous gene by use of a regulatory region of an alcohol oxidase gene (AOD1) (Japanese Patent LOP Publication No. 344,895/1993).
Formate dehydrogenase, like alcohol oxidase, is an enzyme produced in a significant amount, but it is an enzyme located downstream in the methanol metabolism and considered to undergo expression regulation different from that of alcohol oxidase. For example, formate dehydrogenase can, as revealed in the present invention, be induced and expressed depending on culture conditions, even in the presence of glucose by which alcohol oxidase expression is completely inhibited. Therefore, it is expected that a method of expressing a large amount of a heterologous gene, which is different from a method using an alcohol oxidase promoter, can be established.
However, no knowledge of the expression and regulation of formate dehydrogenase from Candida boidinii has been gained up to now. There is a need for a promoter for formate dehydrogenase to elucidate the expression and regulation of said enzyme and to express a heterologous gene efficiently by its strong transcriptional activity.